Can shotgun metagenome approaches effectively target both RNA and DNA viruses in low-viral-load water samples?

Question

[mathavanbioinfo]

Hi, I am working on sewage and river water sample metagenome analysis. With the shotgun metagenome approach, we can obtain genomes of bacterial and archaeal groups. Is it possible to capture the complete genome sequences of RNA viruses specifically? In some cases, the viral load is extremely low in water samples. Is there any way to target both RNA and DNA viruses using the shotgun metagenome? How can we quantify the abundance of viruses (both RNA and DNA viruses) in the metagenome samples? Please share your comments

Answer 1

[theclubstyle]

EDIT: this may not strictly be a bioinformatics question as it seems to be rooted in lab protocols. I'd suggest re-posting on Seqanswers or similar. However:

Theoretically it should be possible using an RNA-seq library prep, but getting accurate relative quantitations from metagenomic assemblages is notoriously difficult, particularly with RNA. Free RNA in water is going to be extremely labile and susceptible to degradation, so you'd likely need to ramp up the PCR cycles in library prep which introduces a lot of bias.

If you're talking about getting DNA and RNA (meta)genomes from the same library, it's a straight no I'm afraid as the library construction is very dependent on the type of molecule you're sequencing; you won't get any RNA information out of a DNA library (apologies if you already knew that). You're probably better off with targeted qPCR for a selection of RNA viruses of interest to be honest.

[mathavanbioinfo]

Thank you for your reply. Our ultimate goal is to study RNA and DNA viruses present in water samples. I understand that they cannot be obtained from the same library. Based on your comment, I will explore the possibilities of targeted qPCR

Answer 2

[joe]

Keep in mind that whatever you purify will be a combination of host DNA/RNA and the viral sequences you're after, so you'll need to remove the 'contamination' of the host, or sequence very deeply.

If you are able to purify the DNA without significant fragmentation, I'd think you can easily purify the viral DNA by size-selection. The viral RNA is more problematic because most of the RNA will be host rRNAs and tRNAs, and then a combination of the viral RNA and host mRNA. Again you might be able to purify the viral RNAs out by size-selection, but unless you known apriori what their sizes will be, you'll likely have a mix of many RNAs. Also the RNA is much more prone to fragmentation compared to DNA. The most important is to remove rRNA - if you know what the host organisms are, a commercially available ribo-depletion kit with custom probes could work. An alternative approach to Illumina-style 'shotgun' RNAseq is to use nanopore DRS, where you could see the entire length of the sequence instead of a fragment (assuming no degradation).

If all you care about is quantification of known viral sequences, qPCR is the way to go.